Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides.

Jordi Bertran-Vicente,Peter Schmieder,Eberhard Krause,Jean-Marc Jeckelmann,Martin Penkert,Olaia Nieto-Garcia,Christian P R Hackenberger

doi:10.1038/ncomms12703

Abstract

In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

Highlights

In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods
Davis and co-workers[30] have developed a two-step method for installing pCys residues on a protein level; this protocol delivers an epimeric mixture of pCys proteins, since it relies on the reaction of dehydroalanine (Dha) with sodium thiophosphate
To show the feasibility of this chemical strategy, our initial experiments were carried out in reactions with naturally occurring cystines using phosphite triesters that had previously been synthesized by our laboratory (3b-e)

Summary

Introduction

In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Important steps have been recently made towards the development of synthetic tools to allow the site-specific phosphorylation of native pLys and pArg peptides and mimetics of pHis in proteins[8,9,10,11,12,13] These chemical tools have permitted the development of mass spectrometry (MS)-based methods or the generation of antibodies, leading to a better understanding of the biological role of these modifications[14,15,16]. Davis and co-workers[30] have developed a two-step method for installing pCys residues on a protein level; this protocol delivers an epimeric mixture of pCys proteins, since it relies on the reaction of dehydroalanine (Dha) with sodium thiophosphate This elegant strategy provided access to pCys residues in proteins, the elimination conditions required to prepare Dha residues and the lack of stereoselectivity may impose certain limitations on the general applicability of this chemical tool in the functional analysis of phosphorylated Cys residues[8]. This work provides a novel synthetic strategy to incorporate native

Methods

Results

Conclusion